Stabilized soluble pre-fusion rsv f polypeptides

ABSTRACT

Stable pre-fusion respiratory syncitial virus (RSV) F polypeptides, immunogenic compositions including the polypeptides, and uses thereof for the prevention and/or treatment of RSV infection are described.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 15/742,265, filed Jan. 5, 2018, which is a Section 371 ofInternational Application No. PCT/EP2016/066104, filed Jul. 7, 2016,which was published in the English language on Jan. 12, 2017 underInternational Publication No. WO 2017/005848 A1, and claims priorityunder 35 U.S.C. § 119(b) to European Application No. 15175654.1, filedJul. 7, 2015, the disclosures of which are incorporated herein byreference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “Sequence_Listing_688097_410U1”, creation date of Oct. 2,2019, and having a size of 44.1 KB. The sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of medicine. The invention inparticular relates to recombinant pre-fusion RSV F polypeptides and usesthereof, e.g. in immunogenic compositions.

BACKGROUND OF THE INVENTION

After discovery of the respiratory syncytial virus (RSV) in the 1950s,the virus soon became a recognized pathogen associated with lower andupper respiratory tract infections in humans. Worldwide, it is estimatedthat 64 million RSV infections occur each year resulting in 160.000deaths (WHO Acute Respiratory Infections Update September 2009). Themost severe disease occurs particularly in premature infants, theelderly and immunocompromised individuals. In children younger than 2years, RSV is the most common respiratory tract pathogen, accounting forapproximately 50% of the hospitalizations due to respiratory infections,and the peak of hospitalization occurs at 2-4 months of age. It has beenreported that almost all children have been infected by RSV by the ageof two. Repeated infection during lifetime is attributed to ineffectivenatural immunity. In the elderly, the RSV disease burden is similar tothose caused by non-pandemic influenza A infections.

RSV is a paramyxovirus, belonging to the subfamily of pneumoviridae. Itsgenome encodes for various proteins, including the membrane proteinsknown as RSV Glycoprotein (G) and RSV fusion (F) protein which are themajor antigenic targets for neutralizing antibodies. Antibodies againstthe fusion-mediating part of the F1 protein can prevent virus uptake inthe cell and thus have a neutralizing effect.

A vaccine against RSV infection is currently not available, but isdesired due to the high disease burden. The RSV fusion glycoprotein (RSVF) is an attractive vaccine antigen since as stated above it is theprincipal target of neutralizing antibodies in human sera. Indeed, aneutralizing monoclonal antibody against RSV F (Palivizumab) can preventsevere disease and has been approved for prophylaxis in infants.

RSV F fuses the viral and host-cell membranes by irreversible proteinrefolding from the labile pre-fusion conformation to the stablepost-fusion conformation. Structures of both conformations have beendetermined for RSV F (McLellan J S, et al. Science 342, 592-598 (2013);McLellan J S, et al. Nat Struct Mol Biol 17, 248-250 (2010); McLellan JS, et al. Science 340, 1113-1117 (2013); Swanson K A, et al. Proceedingsof the National Academy of Sciences of the United States of America 108,9619-9624 (2011)), as well as for the fusion proteins from relatedparamyxoviruses, providing insight into the mechanism of this complexfusion machine. Like other type I fusion proteins, the inactiveprecursor, RSV F₀, requires cleavage during intracellular maturation bya furin-like protease. RSV F contains two furin sites, which leads tothree polypeptides: F2, p27 and F1, with the latter containing ahydrophobic fusion peptide (FP) at its N-terminus. In order to refoldfrom the pre-fusion to the post-fusion conformation, the refoldingregion 1 (RR1) between residue 137 and 216, that includes the FP andheptad repeat A (HRA) has to transform from an assembly of helices,loops and strands to a long continuous helix. The FP, located at theN-terminal segment of RR1, is then able to extend away from the viralmembrane and insert into the proximal membrane of the target cell. Next,the refolding region 2 (RR2), which forms the C-terminal stem in thepre-fusion F spike and includes the heptad repeat B (HRB), relocates tothe other side of the RSV F head and binds the HRA coiled-coil trimerwith the HRB domain to form the six-helix bundle. The formation of theRR1 coiled-coil and relocation of RR2 to complete the six-helix bundleare the most dramatic structural changes that occur during the refoldingprocess.

Most neutralizing antibodies in human sera are directed against thepre-fusion conformation, but due to its instability the pre-fusionconformation has a propensity to prematurely refold into the post-fusionconformation, both in solution and on the surface of the virions. An RSVF protein that has both high expression levels and maintains a stablepre-fusion conformation would be a promising candidate for use in asubunit or vector-based vaccine against RSV.

SUMMARY OF THE INVENTION

The present invention provides stable, recombinant, pre-fusionrespiratory syncytial virus (RSV) fusion (F) polypeptides, i.e.recombinant RSV F polypeptides that are stabilized in the pre-fusionconformation. The RSV F polypeptides of the invention comprise at leastone epitope that is specific to the pre-fusion conformation F protein.In certain embodiments, the pre-fusion RSV F polypeptides are solublepolypeptides. The invention also provides nucleic acid moleculesencoding the pre-fusion RSV F polypeptides according to the inventionand vectors comprising such nucleic acid molecules.

The invention also relates to compositions, preferably immunogeniccompositions, comprising a RSV F polypeptide, a nucleic acid moleculeand/or a vector, and to the use thereof in inducing an immune responseagainst RSV F protein, in particular the use thereof as a vaccine. Theinvention also relates to methods for inducing an anti-respiratorysyncytial virus (RSV) immune response in a subject, comprisingadministering to the subject an effective amount of a pre-fusion RSV Fpolypeptide, a nucleic acid molecule encoding said RSV F polypeptide,and/or a vector comprising said nucleic acid molecule. Preferably, theinduced immune response is characterized by neutralizing antibodies toRSV and/or protective immunity against RSV. In particular aspects, theinvention relates to a method for inducing neutralizing anti-respiratorysyncytial virus (RSV) F protein antibodies in a subject, comprisingadministering to the subject an effective amount of an immunogeniccomposition comprising a pre-fusion RSV F polypeptide, a nucleic acidmolecule encoding said RSV F polypeptide, and/or a vector comprisingsaid nucleic acid molecule.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A: Chromatogram of elution of protein F(A2) KN66EI I76V L203IS215P from cation-exchange column. Blue trace is absorbance at 280 nm,brown trace is conductivity. The arrow indicates the collected peak thatwas eluted at 15% elution step.

FIG. 1B: Superdex200 gel filtration chromatogram of the eluate ofprotein F(A2) KN66EI I76V L203I S215P from the ion-exchange column. Bluetrace is absorbance at 280 nm, brown trace is conductivity. The arrowsindicate the collected peak.

FIG. 2A: SDS-PAGE analysis of the F(A2) KN66EI I76V L203I S215P proteinsample containing peak from the SEC chromatogram under reducing andnon-reducing conditions. F1, F2 fragments in reduced sample and F1+F2 innon-reduced sample are indicated.

FIG. 2B: SDS-PAGE analysis of the F(A2) K66E I76V L203I S215P proteinsample containing peak from the SEC chromatogram under reducing andnon-reducing conditions. F1, F2 fragments in reduced sample and F1+F2 innon-reduced sample are indicated.

FIG. 2C: NativePAGE of F(A2) KN66EI I76V S215P with and without L203Iand F(A2) K66E I76V L203I S215P. Native marker indicates relativeelectrophoretic mobility of the protein but cannot be used fordetermination of protein molecular weight. The position of the bandbetween 242 and 480 kDa corresponds to the position of trimeric RSV Fprotein (indicated by an arrow). The gels are stained with CoomassieBrilliant Blue.

FIG. 3: Additional substitution of Leu 203 to Ile increases pre-fusion Fstability in the metastable pre-fusion F protein with an S215Psubstitution. Pre-fusion RSV F concentrations in cell culturesupernatants were tested on the day of harvest (72 hours posttransfection=day 1) and after storage for 5 and 15 days at 4° C.

FIG. 4: Temperature stability of proteins with L203I. Meltingtemperature (Tm) was determined in purified protein samples by DSF.

FIG. 5: SDS-PAGE analysis of the purified F proteins comprising L203Imutation under reducing (R) and non-reducing (NR) conditions. F1, F2fragments in reduced sample and F1+F2 in non-reduced sample areindicated. The gel was stained with Coomassie Brilliant Blue.

FIG. 6A: SEC-MALS analysis of the purified F proteinF(A2)-KN66EI-I76V-I203L-S215P-D486N, comprising L203I mutation.

FIG. 6B: SEC-MALS analysis of the purified F proteinF(A2)-K66E-I76V-I203L-S215P-D486N, comprising L203I mutation.

DETAILED DESCRIPTION OF THE INVENTION

The fusion protein (F) of the respiratory syncictial virus (RSV) isinvolved in fusion of the viral membrane with a host cell membrane,which is required for viral infection. The RSV F mRNA is translated intoa 574 amino acid precursor protein designated F0, which contains asignal peptide sequence at the N-terminus (e.g. amino acid residues 1-26of SEQ ID NO: 13) that is removed by a signal peptidase in theendoplasmic reticulum. F0 is cleaved at two sites (between amino acidresidues 109/110 and 136/137) by cellular proteases (in particularfurin, or furin-like)) removing a short glycosylated interveningsequence (also referred to a p27 region, comprising the amino acidresidues 110 to 136, and generating two domains or subunits designatedF1 and F2. The F1 domain (amino acid residues 137-574) contains ahydrophobic fusion peptide at its N-terminus and the C-terminus containsthe transmembrane (TM) (amino acid residues 530-550) and cytoplasmicregion (amino acid residues 551-574). The F2 domain (amino acid residues27-109) is covalently linked to F1 by two disulfide bridges. The F1-F2heterodimers are assembled as homotrimers in the virion.

A vaccine against RSV infection is not currently available, but isdesired. One potential approach to producing a vaccine is a subunitvaccine based on purified RSV F protein. However, for this approach itis desirable that the purified RSV F protein is in a conformation whichresembles the conformation of the pre-fusion state of RSV F protein,that is stable over time, and can be produced in sufficient quantities.In addition, for a soluble, subunit-based vaccine, the RSV F proteinneeds to be truncated by deletion of the transmembrane (TM) and thecytoplasmic region to create a soluble secreted F protein (sF). Becausethe TM region is responsible for membrane anchoring and trimerization,the anchorless soluble F protein is considerably more labile than thefull-length protein and will readily refold into the post-fusionend-state. In order to obtain soluble F protein in the stable pre-fusionconformation that shows high expression levels and high stability, thepre-fusion conformation thus needs to be stabilized. Because also thefull length (membrane-bound) RSV F protein is metastable, thestabilization of the pre-fusion conformation is also desirable for anylife attenuated or vector based vaccine approach.

For the stabilization of soluble RSV F, that is cleaved into the F1 andF2 subunit, in the pre-fusion conformation, a fibritin-basedtrimerization domain was fused to the C-terminus of the soluble RSV-FC-terminal end (McLellan et al., Nature Struct. Biol. 17: 2-248-250(2010); McLellan et al., Science 340(6136):1113-7 (2013)). This fibritindomain or ‘Foldon’ is derived from T4 fibritin and was described earlieras an artificial natural trimerization domain (Letarov et al.,Biochemistry Moscow 64: 817-823 (1993); S-Guthe et al., J. Mol. Biol.337: 905-915. (2004)). However, the trimerization domain does not resultin stable pre-fusion RSV-F protein. Moreover, these efforts have not yetresulted in candidates suitable for testing in humans.

Recently, we described combinations of several mutations that arecapable of stabilizing the RSV pre-fusion F conformation (WO2014/174018and WO2014/202570). Thus, stable pre-fusion RSVF polypeptides have beendescribed comprising a mutation of the amino acid residue on position 67and/or a mutation of the amino acid residue on position 215, preferablya mutation of amino acid residue N/T on position 67 into I and/or amutation of amino acid residue S on position 215 into P. In addition,soluble pre-fusion RSV F polypeptides have been described comprising atruncated F1 domain, and at least one stabilizing mutation in the F1and/or F2 domain as compared to the RSV F1 and/or F2 domain in thewild-type RSV F protein, wherein the polypeptide comprises aheterologous trimerization domain linked to said truncated F1 domain.Also further pre-fusion RSV F polypeptides have been described, whereinthe polypeptides comprise at least one further mutation, wherein saidmutation is selected from the group consisting of:

(a) a mutation of the amino acid residue on position 46;

(b) a mutation of the amino acid residue on position 77;

(c) a mutation of the amino acid residue on position 80;

(d) a mutation of the amino acid residue on position 92;

(e) a mutation of the amino acid residue on position 175;

(f) a mutation of the amino acid residue on position 184;

(g) a mutation of the amino acid residue on position 185;

(h) a mutation of the amino acid residue on position 201;

(i) a mutation of the amino acid residue on position 209;

(j) a mutation of the amino acid residue on position 421;

(k) a mutation of the amino acid residue on position 426;

(l) a mutation of the amino acid residue on position 465;

(m) a mutation of the amino acid residue on position 486;

(n) a mutation of the amino acid residue on position 487; and

(o) a mutation of the amino acid residue on position 508.

Preferably, the at least one further mutation is selected from the groupconsisting of:

(a) a mutation of the amino acid residue S on position 46 into G;

(b) a mutation of the amino acid residue K on position 77 into E;

(c) a mutation of the amino acid residue K on position 80 into E;

(d) a mutation of the amino acid residue E on position 92 into D;

(e) a mutation of the amino acid residue N on position 175 into P;

(f) a mutation of the amino acid residue G on position 184 into N;

(g) a mutation of the amino acid residue V on position 185 into N;

(h) a mutation of the amino acid residue K on position 201 into Q;

(i) a mutation of the amino acid residue K on position 209 into Q;

(j) a mutation of the amino acid residue K on position 421 into N;

(k) a mutation of the amino acid residue N on position 426 into S;

(l) a mutation of the amino acid residue K on position 465 into E or Q;

(m) a mutation of the amino acid residue D on position 486 into N;

(n) a mutation of the amino acid residue E on position 487 into Q, N orI; and

(o) a mutation of the amino acid residue K on position 508 into E.

If only one of these mutations was applied (in particular the mutationS215P), a metastable protein was obtained that could be used for theevaluation of the stabilizing effect of alternative substitutions.

According to the present invention it has been found that a mutation ofthe amino acid residue L on position 203 into I further stabilizes theprotein in the pre-fusion conformation.

The present invention thus provides recombinant pre-fusion Fpolypeptides comprising a mutation of the amino acid residue L onposition 203 into I.

The invention in particular provides recombinant pre-fusion respiratorysyncytial virus (RSV) Fusion (F) polypeptides, wherein the polypeptidecomprises at least one, preferably at least two stabilizing mutations inthe F1 and/or F2 domain as compared to the RSV F1 and/or F2 domain in awild-type RSV F protein, wherein at least one of the stabilizingmutations is a mutation of amino acid residue L on position 203 into I.

The invention further provides recombinant pre-fusion F polypeptidescomprising a mutation of the amino acid S on position 215 into P (S215P)and a mutation of the amino acid residue L on position 203 into I.

The present invention thus provides a new stabilization mutation toprovide recombinant stable pre-fusion RSV F polypeptides, i.e. RSV Fpolypeptides that are stabilized in the pre-fusion conformation. Thestable pre-fusion RSV F polypeptides of the invention are in thepre-fusion conformation, i.e. they comprise (display) at least oneepitope that is specific to the pre-fusion conformation F protein. Anepitope that is specific to the pre-fusion conformation F protein is anepitope that is not presented in the post-fusion conformation. Withoutwishing to be bound by any particular theory, it is believed that thepre-fusion conformation of RSV F protein may contain epitopes that arethe same as those on the RSV F protein expressed on natural RSV virions,and therefore may provide advantages for eliciting protectiveneutralizing antibodies.

In certain embodiments, the polypeptides of the invention comprise atleast one epitope that is recognized by a pre-fusion specific monoclonalantibody, comprising a heavy chain CDR1 region of SEQ ID NO: 1, a heavychain CDR2 region of SEQ ID NO: 2, a heavy chain CDR3 region of SEQ IDNO: 3 and a light chain CDR1 region of SEQ ID NO: 4, a light chain CDR2region of SEQ ID NO: 5, and a light chain CDR3 region of SEQ ID NO: 6(hereafter referred to as CR9501) and/or a pre-fusion specificmonoclonal antibody, comprising a heavy chain CDR1 region of SEQ ID NO:7, a heavy chain CDR2 region of SEQ ID NO: 8, a heavy chain CDR3 regionof SEQ ID NO: 9 and a light chain CDR1 region of SEQ ID NO: 10, a lightchain CDR2 region of SEQ ID NO: 11, and a light chain CDR3 region of SEQID NO: 12 (referred to as CR9502). CR9501 and CR9502 comprise the heavyand light chain variable regions, and thus the binding specificities, ofthe antibodies 58C5 and 30D8, respectively, which have previously beenshown to bind specifically to RSV F protein in its pre-fusionconformation and not to the post-fusion conformation (seeWO2012/006596).

In certain embodiments, the recombinant pre-fusion RSV F polypeptidescomprise at least one epitope that is recognized by at least onepre-fusion specific monoclonal antibody as described above and aretrimeric. In certain embodiments, the stable pre-fusion RSV Fpolypeptides according to the invention are soluble and comprise atruncated F1 domain.

It is known that RSV exists as a single serotype having two antigenicsubgroups: A and B. The amino acid sequences of the mature processed Fproteins of the two groups are about 93% identical. As used throughoutthe present application, the amino acid positions are given in referenceto the sequence of RSV F protein from the A2 strain (SEQ ID NO: 13). Asused in the present invention, the wording “the amino acid at position“x” of the RSV F protein thus means the amino acid corresponding to theamino acid at position “x” in the RSV F protein of the RSV A2 strain ofSEQ ID NO: 13. Note that, in the numbering system used throughout thisapplication 1 refers to the N-terminal amino acid of an immature F0protein (SEQ ID NO: 13) When a RSV strain other than the A2 strain isused, the amino acid positions of the F protein are to be numbered withreference to the numbering of the F protein of the A2 strain of SEQ IDNO: 1 by aligning the sequences of the other RSV strain with the Fprotein of SEQ ID NO: 13 with the insertion of gaps as needed. Sequencealignments can be done using methods well known in the art, e.g. byCLUSTALW, Bioedit or CLC Workbench.

An amino acid according to the invention can be any of the twentynaturally occurring (or ‘standard’ amino acids) or variants thereof,such as e.g. D-amino acids (the D-enantiomers of amino acids with achiral center), or any variants that are not naturally found inproteins, such as e.g. norleucine. The standard amino acids can bedivided into several groups based on their properties. Important factorsare charge, hydrophilicity or hydrophobicity, size and functionalgroups. These properties are important for protein structure andprotein-protein interactions. Some amino acids have special propertiessuch as cysteine, that can form covalent disulfide bonds (or disulfidebridges) to other cysteine residues, proline that induces turns of thepolypeptide backbone, and glycine that is more flexible than other aminoacids. Table 1 shows the abbreviations and properties of the standardamino acids.

It will be appreciated by a skilled person that the mutations can bemade to the protein by routine molecular biology procedures. Themutations according to the invention preferably result in increasedexpression levels and/or increased stabilization of the pre-fusion RSV Fpolypeptides as compared RSV F polypeptides that do not comprise thesemutation(s).

In certain embodiments, the pre-fusion RSV F polypeptides are soluble.

Thus, in certain embodiments, the pre-fusion RSV F polypeptides comprisea truncated F1 domain, wherein polypeptide comprises at least onestabilizing mutation in the F1 and/or F2 domain as compared to the RSVF1 and/or F2 domain in the wild-type RSV F protein. In certainembodiments, the soluble pre-fusion RSV F polypeptides further comprisea heterologous trimerization domain linked to said truncated F1 domain.According to the invention, it was shown that by linking a heterologoustrimerization domain to the C-terminal amino acid residue of a truncatedF1 domain, combined with the stabilizing mutation(s), RSV F polypeptidesare provided that show high expression and that bind topre-fusion-specific antibodies, indicating that the polypeptides are inthe pre-fusion conformation. In addition, the RSV F polypeptides arestabilized in the pre-fusion conformation, i.e. even after processing ofthe polypeptides they still bind to the pre-fusion specific antibodiesCR9501 and/or CR9502, indicating that the pre-fusion specific epitope isretained.

In certain embodiments, the pre-fusion RSV F polypeptides comprise atleast three mutations (as compared to a wild-type RV F protein).

In certain embodiments, the pre-fusion RSV F polypeptides comprise atleast one further mutation selected from the group consisting of:

(a) a mutation of the amino acid residue on position 46;

(b) a mutation of the amino acid residue on position 67;

(c) a mutation of the amino acid residue on position 83;

(d) a mutation of the amino acid residue on position 92;

(e) a mutation of the amino acid residue on position 184;

(f) a mutation of the amino acid residue on position 207;

(g) a mutation of the amino acid residue on position 486; and

(h) a mutation of the amino acid residue on position 487.

In certain embodiments, the at least one further mutation is selectedfrom the group consisting of:

(a) a mutation of the amino acid residue S on position 46 into G;

(b) a mutation of the amino acid residue N/T on position 67 into I;

(c) a mutation of the amino acid residue L on position 83 into M:

(d) a mutation of the amino acid residue E on position 92 into D;

(e) a mutation of the amino acid residue G on position 184 into N;

(f) a mutation of the amino acid residue V on position 207 into I:

(g) a mutation of the amino acid residue D on position 486 into N; and

(h) a mutation of the amino acid residue E on position 487 into Q, N orI.

In certain embodiments, the polypeptides comprise at least twomutations.

In certain embodiments, the polypeptides comprise at least threemutations.

In certain embodiments, the polypeptides comprise at least four, five orsix mutations.

In certain other embodiments, the heterologous trimerization domaincomprises the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ IDNO: 14).

As described above, in certain embodiments, the polypeptides of theinvention comprise a truncated F1 domain. As used herein a “truncated”F1 domain refers to a F1 domain that is not a full length F1 domain,i.e. wherein either N-terminally or C-terminally one or more amino acidresidues have been deleted. According to the invention, at least thetransmembrane domain and cytoplasmic tail have been deleted to permitexpression as a soluble ectodomain.

In certain other embodiments, the trimerization domain is linked toamino acid residue 513 of the RSV F1 domain. In certain embodiments, thetrimerization domain comprises SEQ ID NO: 14 and is linked to amino acidresidue 513 of the RSV F1 domain.

In certain embodiments, the F1 domain and/or the F domain are from anRSV A strain. In certain embodiments the F1 and/or F2 domain are fromthe RSV A2 strain of SEQ ID NO: 13.

In certain embodiments, the F1 domain and/or the F domain are from anRSV B strain. In certain embodiments the F1 and/or F2 domain are fromthe RSV B strain of SEQ ID NO: 15.

In certain embodiments, the F1 domain and/or the F domain are from anRSV CL57-v244 strain. In certain embodiments the F1 and/or F2 domain arefrom the RSV CL57-v244 strain of SEQ ID NO: 22.

In certain embodiments, the F1 and F2 domain are from the same RSVstrain. In certain embodiments, the pre-fusion RSV F polypeptides arechimeric polypeptides, i.e. comprising F1 and F2 domains that are fromdifferent RSV strains.

In certain embodiments, the pre-fusion RSV F polypeptides comprise anamino acid sequence selected from the group consisting of SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO: 24.

In certain embodiments, the level of expression of the pre-fusion RSV Fpolypeptides of the invention is increased, as compared to a wild-typeRSV F polypeptide. In certain embodiments the level of expression isincreased at least 5-fold, preferably up to 10-fold. In certainembodiments, the level of expression is increased more than 10-fold.

The pre-fusion RSV F polypeptides according to the invention are stable,i.e. do not readily change into the post-fusion conformation uponprocessing of the polypeptides, such as e.g. purification, freeze-thawcycles, and/or storage etc.

In certain embodiments, the pre-fusion RSV F polypeptides according tothe invention have an increased stability upon storage a 4° C. ascompared to a RSV F polypeptide without the mutation(s). In certainembodiments, the polypeptides are stable upon storage at 4° C. for atleast 30 days, preferably at least 60 days, preferably at least 6months, even more preferably at least 1 year. With “stable uponstorage”, it is meant that the polypeptides still display the at leastone epitope specific for the a pre-fusion specific antibody (e.g.CR9501) upon storage of the polypeptide in solution (e.g. culturemedium) at 4° C. for at least 30 days, e.g. as determined using a methodas described in Example 7 or 9. In certain embodiments, the polypeptidesdisplay the at least one pre-fusion specific epitope for at least 6months, preferably for at least 1 year upon storage of the pre-fusionRSV F polypeptides at 4° C.

In certain embodiments, the pre-fusion RSV F polypeptides according tothe invention have an increased stability when subjected to heat, ascompared to RSV F polypeptides without said mutation(s). In certainembodiments, the pre-fusion REV F polypeptides are heat stable for atleast 30 minutes at a temperature of 55° C., preferably at 58° C., morepreferably at 60° C. With “heat stable” it is meant that thepolypeptides still display the at least one pe-fusion specific epitopeafter having been subjected for at least 30 minutes to an increasedtemperature (i.e. a temperature of 55° C. or above), e.g. as determinedusing a method as described in Example 6.

In certain embodiments, the polypeptides display the at least onepre-fusion specific epitope after being subjected to 1 to 6 freeze-thawcycles in an appropriate formulation buffer.

As used throughout the present application nucleotide sequences areprovided from 5′ to 3′ direction, and amino acid sequences fromN-terminus to C-terminus, as custom in the art.

In certain embodiments, the encoded polypeptides according to theinvention further comprise a leader sequence, also referred to as signalsequence or signal peptide, corresponding to amino acids 1-26 of SEQ IDNO: 13. This is a short (typically 5-30 amino acids long) peptidepresent at the N-terminus of the majority of newly synthesized proteinsthat are destined towards the secretory pathway. In certain embodiments,the polypeptides according to the invention do not comprise a leadersequence.

In certain embodiments, the polypeptides comprise a HIS-Tag. A His-Tagor polyhistidine-tag is an amino acid motif in proteins that consists ofat least five histidine (H) residues, often at the N- or C-terminus ofthe protein, which is generally used for purification purposes.

The present invention further provides nucleic acid molecules encodingthe RSV F polypeptides according to the invention.

In preferred embodiments, the nucleic acid molecules encoding thepolypeptides according to the invention are codon-optimized forexpression in mammalian cells, preferably human cells. Methods ofcodon-optimization are known and have been described previously (e.g. WO96/09378). A sequence is considered codon-optimized if at least onenon-preferred codon as compared to a wild type sequence is replaced by acodon that is more preferred. Herein, a non-preferred codon is a codonthat is used less frequently in an organism than another codon codingfor the same amino acid, and a codon that is more preferred is a codonthat is used more frequently in an organism than a non-preferred codon.The frequency of codon usage for a specific organism can be found incodon frequency tables, such as in http://www.kazusa.or.jp/codon.Preferably more than one non-preferred codon, preferably most or allnon-preferred codons, are replaced by codons that are more preferred.Preferably the most frequently used codons in an organism are used in acodon-optimized sequence. Replacement by preferred codons generallyleads to higher expression.

It will be understood by a skilled person that numerous differentpolynucleotides and nucleic acid molecules can encode the samepolypeptide as a result of the degeneracy of the genetic code. It isalso understood that skilled persons may, using routine techniques, makenucleotide substitutions that do not affect the polypeptide sequenceencoded by the nucleic acid molecules to reflect the codon usage of anyparticular host organism in which the polypeptides are to be expressed.Therefore, unless otherwise specified, a “nucleotide sequence encodingan amino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may or maynot include introns.

Nucleic acid sequences can be cloned using routine molecular biologytechniques, or generated de novo by DNA synthesis, which can beperformed using routine procedures by service companies having businessin the field of DNA synthesis and/or molecular cloning (e.g. GeneArt,GenScripts, Invitrogen, Eurofins).

The invention also provides vectors comprising a nucleic acid moleculeas described above. In certain embodiments, a nucleic acid moleculeaccording to the invention thus is part of a vector. Such vectors caneasily be manipulated by methods well known to the person skilled in theart, and can for instance be designed for being capable of replicationin prokaryotic and/or eukaryotic cells. In addition, many vectors can beused for transformation of eukaryotic cells and will integrate in wholeor in part into the genome of such cells, resulting in stable host cellscomprising the desired nucleic acid in their genome. The vector used canbe any vector that is suitable for cloning DNA and that can be used fortranscription of a nucleic acid of interest. Suitable vectors accordingto the invention are e.g. adenovectors, alphavirus, paramyxovirus,vaccinia virus, herpes virus, retroviral vectors etc. The person skilledin the art is capable of choosing suitable expression vectors, andinserting the nucleic acid sequences of the invention in a functionalmanner.

Host cells comprising the nucleic acid molecules encoding the pre-fusionRSV F polypeptides form also part of the invention. The pre-fusion RSV Fpolypeptides may be produced through recombinant DNA technologyinvolving expression of the molecules in host cells, e.g. Chinesehamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell linessuch as HEK293 cells, PER.C6 cells, or yeast, fungi, insect cells, andthe like, or transgenic animals or plants. In certain embodiments, thecells are from a multicellular organism, in certain embodiments they areof vertebrate or invertebrate origin. In certain embodiments, the cellsare mammalian cells. In certain embodiments, the cells are human cells.In general, the production of a recombinant proteins, such thepre-fusion RSV F polypeptides of the invention, in a host cell comprisesthe introduction of a heterologous nucleic acid molecule encoding thepolypeptide in expressible format into the host cell, culturing thecells under conditions conducive to expression of the nucleic acidmolecule and allowing expression of the polypeptide in said cell. Thenucleic acid molecule encoding a protein in expressible format may be inthe form of an expression cassette, and usually requires sequencescapable of bringing about expression of the nucleic acid, such asenhancer(s), promoter, polyadenylation signal, and the like. The personskilled in the art is aware that various promoters can be used to obtainexpression of a gene in host cells. Promoters can be constitutive orregulated, and can be obtained from various sources, including viruses,prokaryotic, or eukaryotic sources, or artificially designed.

Cell culture media are available from various vendors, and a suitablemedium can be routinely chosen for a host cell to express the protein ofinterest, here the pre-fusion RSV F polypeptides. The suitable mediummay or may not contain serum.

A “heterologous nucleic acid molecule” (also referred to herein as‘transgene’) is a nucleic acid molecule that is not naturally present inthe host cell. It is introduced into for instance a vector by standardmolecular biology techniques. A transgene is generally operably linkedto expression control sequences. This can for instance be done byplacing the nucleic acid encoding the transgene(s) under the control ofa promoter. Further regulatory sequences may be added. Many promoterscan be used for expression of a transgene(s), and are known to theskilled person, e.g. these may comprise viral, mammalian, syntheticpromoters, and the like. A non-limiting example of a suitable promoterfor obtaining expression in eukaryotic cells is a CMV-promoter (U.S.Pat. No. 5,385,839), e.g. the CMV immediate early promoter, for instancecomprising nt. −735 to +95 from the CMV immediate early geneenhancer/promoter. A polyadenylation signal, for example the bovinegrowth hormone polyA signal (U.S. Pat. No. 5,122,458), may be presentbehind the transgene(s). Alternatively, several widely used expressionvectors are available in the art and from commercial sources, e.g. thepcDNA and pEF vector series of Invitrogen, pMSCV and pTK-Hyg from BDSciences, pCMV-Script from Stratagene, etc, which can be used torecombinantly express the protein of interest, or to obtain suitablepromoters and/or transcription terminator sequences, polyA sequences,and the like.

The cell culture can be any type of cell culture, including adherentcell culture, e.g. cells attached to the surface of a culture vessel orto microcarriers, as well as suspension culture. Most large-scalesuspension cultures are operated as batch or fed-batch processes becausethey are the most straightforward to operate and scale up. Nowadays,continuous processes based on perfusion principles are becoming morecommon and are also suitable. Suitable culture media are also well knownto the skilled person and can generally be obtained from commercialsources in large quantities, or custom-made according to standardprotocols. Culturing can be done for instance in dishes, roller bottlesor in bioreactors, using batch, fed-batch, continuous systems and thelike. Suitable conditions for culturing cells are known (see e.g. TissueCulture, Academic Press, Kruse and Paterson, editors (1973), and R. I.Freshney, Culture of animal cells: A manual of basic technique, fourthedition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9)).

The invention further provides compositions comprising a pre-fusion RSVF polypeptide and/or a nucleic acid molecule, and/or a vector, asdescribed above. The invention thus provides compositions comprising apre-fusion RSV F polypeptide that displays an epitope that is present ina pre-fusion conformation of the RSV F protein but is absent in thepost-fusion conformation. The invention also provides compositionscomprising a nucleic acid molecule and/or a vector, encoding suchpre-fusion RSV F polypeptide. The invention further provides immunogeniccompositions comprising a pre-fusion RSV F polypeptide, and/or a nucleicacid molecule, and/or a vector, as described above. The invention alsoprovides the use of a stabilized pre-fusion RSV F polypeptide, a nucleicacid molecule, and/or a vector, according to the invention, for inducingan immune response against RSV F protein in a subject. Further providedare methods for inducing an immune response against RSV F protein in asubject, comprising administering to the subject a pre-fusion RSV Fpolypeptide, and/or a nucleic acid molecule, and/or a vector accordingto the invention. Also provided are pre-fusion RSV F polypeptides,nucleic acid molecules, and/or vectors, according to the invention foruse in inducing an immune response against RSV F protein in a subject.Further provided is the use of the pre-fusion RSV F polypeptides, and/ornucleic acid molecules, and/or vectors according to the invention forthe manufacture of a medicament for use in inducing an immune responseagainst RSV F protein in a subject.

The pre-fusion RSV F polypeptides, nucleic acid molecules, or vectors ofthe invention may be used for prevention (prophylaxis) and/or treatmentof RSV infections. In certain embodiments, the prevention and/ortreatment may be targeted at patient groups that are susceptible RSVinfection. Such patient groups include, but are not limited to e.g., theelderly (e.g. ≥50 years old, ≥60 years old, and preferably ≥65 yearsold), the young (e.g. ≤5 years old, ≤1 year old), hospitalized patientsand patients who have been treated with an antiviral compound but haveshown an inadequate antiviral response.

The pre-fusion RSV F polypeptides, nucleic acid molecules and/or vectorsaccording to the invention may be used e.g. in stand-alone treatmentand/or prophylaxis of a disease or condition caused by RSV, or incombination with other prophylactic and/or therapeutic treatments, suchas (existing or future) vaccines, antiviral agents and/or monoclonalantibodies.

The invention further provides methods for preventing and/or treatingRSV infection in a subject utilizing the pre-fusion RSV F polypeptides,nucleic acid molecules and/or vectors according to the invention. In aspecific embodiment, a method for preventing and/or treating RSVinfection in a subject comprises administering to a subject in needthereof an effective amount of a pre-fusion RSV F polypeptide, nucleicacid molecule and/or a vector, as described above. A therapeuticallyeffective amount refers to an amount of a polypeptide, nucleic acidmolecule or vector, that is effective for preventing, amelioratingand/or treating a disease or condition resulting from infection by RSV.Prevention encompasses inhibiting or reducing the spread of RSV orinhibiting or reducing the onset, development or progression of one ormore of the symptoms associated with infection by RSV. Amelioration asused in herein may refer to the reduction of visible or perceptibledisease symptoms, viremia, or any other measurable manifestation ofinfluenza infection.

For administering to subjects, such as humans, the invention may employpharmaceutical compositions comprising a pre-fusion RSV F polypeptide, anucleic acid molecule and/or a vector as described herein, and apharmaceutically acceptable carrier or excipient. In the presentcontext, the term “pharmaceutically acceptable” means that the carrieror excipient, at the dosages and concentrations employed, will not causeany unwanted or harmful effects in the subjects to which they areadministered. Such pharmaceutically acceptable carriers and excipientsare well known in the art (see Remington's Pharmaceutical Sciences, 18thedition, A. R. Gennaro, Ed., Mack Publishing Company [1990];Pharmaceutical Formulation Development of Peptides and Proteins, S.Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook ofPharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., PharmaceuticalPress [2000]). The RSV F polypeptides, or nucleic acid molecules,preferably are formulated and administered as a sterile solutionalthough it may also be possible to utilize lyophilized preparations.Sterile solutions are prepared by sterile filtration or by other methodsknown per se in the art. The solutions are then lyophilized or filledinto pharmaceutical dosage containers. The pH of the solution generallyis in the range of pH 3.0 to 9.5, e.g. pH 5.0 to 7.5. The RSV Fpolypeptides typically are in a solution having a suitablepharmaceutically acceptable buffer, and the composition may also containa salt. Optionally stabilizing agent may be present, such as albumin. Incertain embodiments, detergent is added. In certain embodiments, the RSVF polypeptides may be formulated into an injectable preparation.

In certain embodiments, a composition according to the invention furthercomprises one or more adjuvants. Adjuvants are known in the art tofurther increase the immune response to an applied antigenicdeterminant. The terms “adjuvant” and “immune stimulant” are usedinterchangeably herein, and are defined as one or more substances thatcause stimulation of the immune system. In this context, an adjuvant isused to enhance an immune response to the RSV F polypeptides of theinvention. Examples of suitable adjuvants include aluminium salts suchas aluminium hydroxide and/or aluminium phosphate; oil-emulsioncompositions (or oil-in-water compositions), including squalene-wateremulsions, such as MF59 (see e.g. WO 90/14837); saponin formulations,such as for example QS21 and Immunostimulating Complexes (ISCOMS) (seee.g. U.S. Pat. No. 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762,WO 2005/002620); bacterial or microbial derivatives, examples of whichare monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motifcontaining oligonucleotides, ADP-ribosylating bacterial toxins ormutants thereof, such as E. coli heat labile enterotoxin LT, choleratoxin CT, and the like; eukaryotic proteins (e.g. antibodies orfragments thereof (e.g. directed against the antigen itself or CD1a,CD3, CD7, CD80) and ligands to receptors (e.g. CD40L, GMCSF, GCSF, etc),which stimulate immune response upon interaction with recipient cells.In certain embodiments the compositions of the invention comprisealuminium as an adjuvant, e.g. in the form of aluminium hydroxide,aluminium phosphate, aluminium potassium phosphate, or combinationsthereof, in concentrations of 0.05-5 mg, e.g. from 0.075-1.0 mg, ofaluminium content per dose.

The pre-fusion RSV F polypeptides may also be administered incombination with or conjugated to nanoparticles, such as e.g. polymers,liposomes, virosomes, virus-like particles. The pre-fusion Fpolypeptides may be combined with, encapsidated in or conjugated to thenanoparticles with or without adjuvant. Encapsulation within liposomesis described, e.g. in U.S. Pat. No. 4,235,877. Conjugation tomacromolecules is disclosed, for example in U.S. Pat. Nos. 4,372,945 or4,474,757.

In other embodiments, the compositions do not comprise adjuvants.

In certain embodiments, the invention provides methods for making avaccine against respiratory syncytial virus (RSV), comprising providinga composition according to the invention and formulating it into apharmaceutically acceptable composition. The term “vaccine” refers to anagent or composition containing an active component effective to inducea certain degree of immunity in a subject against a certain pathogen ordisease, which will result in at least a decrease (up to completeabsence) of the severity, duration or other manifestation of symptomsassociated with infection by the pathogen or the disease. In the presentinvention, the vaccine comprises an effective amount of a pre-fusion RSVF polypeptide and/or a nucleic acid molecule encoding a pre-fusion RSV Fpolypeptide, and/or a vector comprising said nucleic acid molecule,which results in an immune response against the F protein of RSV. Thisprovides a method of preventing serious lower respiratory tract diseaseleading to hospitalization and the decrease in frequency ofcomplications such as pneumonia and bronchiolitis due to RSV infectionand replication in a subject. The term “vaccine” according to theinvention implies that it is a pharmaceutical composition, and thustypically includes a pharmaceutically acceptable diluent, carrier orexcipient. It may or may not comprise further active ingredients. Incertain embodiments it may be a combination vaccine that furthercomprises other components that induce an immune response, e.g. againstother proteins of RSV and/or against other infectious agents. Theadministration of further active components may for instance be done byseparate administration or by administering combination products of thevaccines of the invention and the further active components.

Compositions may be administered to a subject, e.g. a human subject. Thetotal dose of the RSV F polypeptides in a composition for a singleadministration can for instance be about 0.01 μg to about 10 mg, e.g. 1μg-1 mg, e.g. 10 μg-100 μg. Determining the recommended dose will becarried out by experimentation and is routine for those skilled in theart.

Administration of the compositions according to the invention can beperformed using standard routes of administration. Non-limitingembodiments include parenteral administration, such as intradermal,intramuscular, subcutaneous, transcutaneous, or mucosal administration,e.g. intranasal, oral, and the like. In one embodiment a composition isadministered by intramuscular injection. The skilled person knows thevarious possibilities to administer a composition, e.g. a vaccine inorder to induce an immune response to the antigen(s) in the vaccine.

A subject as used herein preferably is a mammal, for instance a rodent,e.g. a mouse, a cotton rat, or a non-human-primate, or a human.Preferably, the subject is a human subject.

The polypeptides, nucleic acid molecules, vectors, and/or compositionsmay also be administered, either as prime, or as boost, in a homologousor heterologous prime-boost regimen. If a boosting vaccination isperformed, typically, such a boosting vaccination will be administeredto the same subject at a time between one week and one year, preferablybetween two weeks and four months, after administering the compositionto the subject for the first time (which is in such cases referred to as‘priming vaccination’). In certain embodiments, the administrationcomprises a prime and at least one booster administration.

In addition, the polypeptides of the invention may be used as diagnostictool, for example to test the immune status of an individual byestablishing whether there are antibodies in the serum of suchindividual capable of binding to the polypeptide of the invention. Theinvention thus also relates to an in vitro diagnostic method fordetecting the presence of an RSV infection in a patient said methodcomprising the steps of a) contacting a biological sample obtained fromsaid patient with a polypeptide according to the invention; and b)detecting the presence of antibody-polypeptide complexes.

The invention further provides a method for stabilizing the pre-fusionconformation of an RSV F polypeptide, comprising introducing at leastone mutation in a RSV F1 and/or F2 domain, as compared to a wild-typeRSV F1 and/or F2 domain, wherein the at least one mutation locks thealpha helix 4 from hinging or moving by a mutation in the triple helixin the RSV apex (alpha helix 1, 4 and 5). In certain embodiments, the atleast one mutation in alpha 4 is a mutation of the amino acid residueLeu into Ile at position 203.

Stabilized pre-fusion RSV F polypeptides obtainable and/or obtained bysuch method also form part of the invention, as well as uses thereof asdescribed above.

EXAMPLES Example 1

In order to stabilize the labile apex of the pre-fusion conformation ofRSV F, a Leu203Ile substitution in alpha4 was introduced in a metastableRSV F variant with a stabilizing S215P mutation and a C-terminalfibritin motif.

Expression and Purification of RSV F Protein

Recombinant proteins were expressed in 293 Freestyle cells (LifeTechnologies). The cells were transiently transfected using 293Fectin(Life Technologies) according to the manufacturer's instructions andcultured in a shaking incubator at 37° C. and 10% CO₂. The culturesupernatants containing F protein were harvested on the 5^(th) day aftertransfection. Sterile-filtered supernatants were stored at 4° C. untiluse. The recombinant polypeptides were purified by a 2-step protocolapplying a cation-exchange chromatography followed by size-exclusionchromatography (FIG. 1). For the ion-exchange step the culturesupernatant was diluted with 2 volumes of 50 mM NaOAc pH 5.0 and passedover a 5 ml HiTrap Capto S (GE Healthcare) column at 5 ml per minute.Subsequently the column was washed with 10 column volumes (CV) of 20 mMNaOAc, 50 mM NaCl, 0.01% (v/v) tween20, pH 5 and eluted with 15% stepelution of 50 mM NaOAc, 1 M NaCl, 0.01% (v/v) tween20, pH 5. The eluatewas concentrated and the protein was further purified on a Superdex200column (GE Healthcare) using 40 mM Tris, 150 mM NaCl, 0.01% (v/v)tween20, and pH 7.4 as running buffer. The purified protein was analyzedon SDS-PAGE and Native PAGE (FIG. 2). Proteins were visualized on thegel upon staining with Coomassie Brilliant Blue. Main bands on theSDS-PAGE are corresponding to F1 and F2 domains of RSV F protein inreduced samples; in non-reduces samples the main band corresponds to thesize of F1+F2 domains linked together with disulfide bonds. On theNative Page electrophoretic mobility of the protein corresponds to oneof the RSV F trimer. The pre-fusion conformation of the purified proteinwas confirmed by binding to CR9501 antibody (data not shown). Thepurified pre-fusion trimeric RSV F protein was stored at 4 C untilfurther analysis.

Stability Studies

The ability of the pre-fusion protein to spontaneously convert intopost-fusion conformation was assessed in a storage stability assay. Thecrude cell culture supernatant samples were stored at 4° C. andconcentration of the F protein in the samples was measured on Octetinstrument by quantitative assay as described above. The measurement wasdone on the day of supernatant harvest (day 1) and after storage forindicated period of time. CR9501 is a monoclonal antibody that onlyrecognizes the pre-fusion F conformation (WO2012/006596) and was used tomeasure prefusion RSV F protein concentration.

Temperature stability of the purified proteins was determined bydifferential scanning fluorometry (DSF). The purified pre-fusion Fprotein was mixed with SYPRO orange fluorescent dye (Life TechnologiesS6650) in a 96-well optical qPCR plate. The optimal dye and proteinconcentration was determined experimentally (data not shown). Allprotein dilutions were performed in PBS, and a negative control samplecontaining the dye only was used as a reference subtraction. Themeasurement was performed in a qPCR instrument (Applied Biosystems ViiA7) using the following parameters: a temperature ramp from 25-95° C.with a rate of 0.015° C. per second. Data was collected continuously.The 1^(st) derivative of the melting curves was plotted using GraphPadPRISM software (version 5.04). Melting temperatures were calculated atthe minimum point of the derivative curve.

As shown in FIG. 3 the metastable F variant that was only stabilizedwith the S215P substitution lost almost all binding to thepre-fusion-specific Mab CR9501 after storage for 5 days at 4° C. Incontrast, the additional L203I substitution increased the stability ofthis metastable RSV F variant dramatically. Total binding of CR9501binding at day of harvest was higher and only a small drop in CR9501binding was observed after storage of 15 days at 4° C. An additionalsubstitution of Leu 203 to Ile increases pre-fusion F stability storagestability compared to the metastable pre-fusion F protein that onlycontained the single S215P substitution. The additional L203Isubstitution also increased the heat stability (59.5° C.) compared tothe variant that contained the N67I and S215P substitution (57.0° C.)according to the DSF experiments (FIG. 4).

The constructs were tested for expression levels, storage stability andantibody binding with the antibody CR9501, specific for the pre-fusionconformation of RSV-F. The amino acid sequences of the heavy and lightchain variable regions, and of the heavy and light chain CDRs of thisantibody are given below. CR9501 comprises the binding regions of theantibodies referred to as 58C5 in WO2012/006596.

Example 2

According to the present invention, it has been shown that thecombination of the Leu203Ile substitution with other stabilizingmutations results in a very stable RSV F protein.

Expression, Purification and Characterization of RSV F Protein

Recombinant proteins were expressed and purified as described above. Thepurified protein was analyzed on SDS-PAGE (FIG. 5). Proteins were purewith the only bands observed on gels corresponding to F1 and F2 domainsof RSV F protein in reduced samples and to F1+F2 domains linked togetherin non-reduces samples. SEC-MALS analysis of the purified samples (FIG.6) demonstrated that the only protein species present in the sample hadmolecular weight of ˜170 kDa which corresponds to expected molecularweight of glycosylated RSV F trimer. The SEC-MALS was performed on anAgilent HPLC system using a TSK G3000SWXL column (Tosoh Bioscience).MALS measurements were performed using a MiniDAWN Treos in-line detector(Wyatt Technology). Protein concentration was monitored using a UVmonitor at 280 nm and a refractive index detector at 660 nm (OptilabTrEX, Wyatt Technology). The detectors were plumbed in the order: UV,MALS, RI. SEC-MALS experiments employed MALS buffer (17.3 grNa2HPO4*2H2O/L, 7.3 gr NaH2PO4*H2O/L, 2.9 gr NaCl/L, pH 7) and a flowrate of 1 ml/min. Data were analyzed using Astra software 6.1 (WyattTechnology) using the refractive index detector and a refractive indexincrement (dn/dc) value of 0.141 ml/g. The molecular weight isdetermined by using the peak maximum of the refractive index results,the total peak area is determined using the total peak area % of theUV-signal corrected for the extinction coefficient.

Stability Studies

Temperature stability of the purified proteins was determined bydifferential scanning fluorometry (DSF) as described in Example 1. Asshown in FIG. 4, when the L203I substitution was combined with S215P,D486N with or without N67I the heat stability of the proteins increaseto ˜67 and ˜70° C. respectively.

TABLE 1 Standard amino acids, abbreviations and properties Side chainSide chain Amino Acid 3-Letter 1-Letter polarity charge (pH 7.4) alanineAla A non-polar Neutral arginine Arg R polar Positive asparagine Asn Npolar Neutral aspartic acid Asp D polar Negative cysteine Cys Cnon-polar Neutral glutamic acid Glu E polar Negative glutamine Gln Qpolar Neutral glycine Gly G non-polar Neutral histidine His H polarpositive (10%) neutral (90%) isoleucine Ile I non-polar Neutral leucineLeu L non-polar Neutral lysine Lys K polar Positive methionine Met Mnon-polar Neutral phenylalanine Phe F non-polar Neutral proline Pro Pnon-polar Neutral serine Ser S polar Neutral threonine Thr T polarNeutral tryptophan Trp W non-polar Neutral tyrosine Tyr Y polar Neutralvaline Val V non-polar Neutral

TABLE 2 Amino acid sequences of antibodies CR9501 and CR9502 AbVH domain VH CDR1 VH CDR2 VH CDR3 CR9501 Amino acids 1- GASINSDNYYWTHISYTGNTYYTPSLKS CGAYVLISNCGWFDS 125 of SEQ ID (SEQ ID NO: 1)(SEQ ID NO: 2) (SEQ ID NO: 3) NO: 16 CR9502 Amino acids 1- GFTFSGHTIAWVSTNNGNTEYAQKI EWLVMGGFAFDH 121 of SEQ ID (SEQ ID NO: 7) QG(SEQ ID NO: 9) NO: 18 (SEQ ID NO: 8) Ab VL domain VL CDR1 VL CDR2VL CDR3 CR9501 Amino acids 1-107 QASQDISTYLN GASNLET QQYQYLPYTof SEQ ID NO: 17 (SEQ ID NO: 4) (SEQ ID NO: 5) (SEQ ID NO: 6) CR9502Amino acids 1-110 GANNIGSQNVH DDRDRPS QVWDSSRDQAVI of SEQ ID NO: 19(SEQ ID NO: 10) (SEQ ID NO: 11) (SEQ ID NO: 12)

The amino acid sequence of several of the pre-fusion RSV F constructs isgiven below. It is noted that the amino acid numbering in the differentconstructs described herein is based on the wild-type sequence (SEQ IDNO: 13 with two modifications (K66E and I76V).

Sequences RSV F protein A2 full length sequence (SEQ ID NO: 13)MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN RSV F protein B1 full length sequence (SEQ IDNO: 15) MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITTIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK SEQ ID NO: 14 (fibritin)GYIPEAPRDGQAYVRKDGEWVLLSTFL FA2, K66E, I76V, S215P (SEQ ID NO: 20)MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLFA2, K66E, I76V, L203I, S215P (SEQ ID NO: 21)MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQILPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLFA2, K66E, I76V, L203I, S215P, D486N (SEQ ID NO: 23)MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQILPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSNEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLFA2, K66E, N67I, I76V, L203I, S215P, D486N (SEQ ID NO: 24)MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQILPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSNEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLRSV F protein CL57-v224 full length sequence (SEQ ID NO: 22)MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANNRARRELPRFMNYTLNNTKNNNVTLSKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNVGKSTTNIMITTIIIVIIVILLLLIAVGLFLYCKARSTPVTLSKDQLSGINNIAFSN CR9501 heavy chain (SEQ ID NO: 16):QVQLVQSGPGLVKPSQTLALTCNVSGASINSDNYYWTWIRQRPGGGLEWIGHISYTGNTYYTPSLKSRLSMSLETSQSQFSLRLTSVTAADSAVYFCAACGAYVLISNCGWFDSWGQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC CR9501 light chain (SEQ ID NO: 17):EIVMTQSPSSLSASIGDRVTITCQASQDISTYLNWYQQKPGQAPRLLIYGASNLETGVPSRFTGSGYGTDFSVTISSLQPEDIATYYCQQYQYLPYTFAPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECCR9502 heavy chain (SEQ ID NO: 18):EVQLLQSGAELKKPGASVKISCKTSGFTF SGHTIAWVRQAPGQGLEWMGWVSTNNGNTEYAQKIQGRVTMTMDTSTSTVYMELRSLTSDDTAVYFCAREWLVMGGFAFDHWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC CR9502 light chain (SEQ ID NO: 19):QSVLTQASSVSVAPGQTARITCGANNIGSQNVHWYQQKPGQAPVLVVYDDRDRPSGIPDRFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSRDQAVIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTIAPTECS

1. A nucleic acid molecule encoding a recombinant pre-fusion respiratorysyncytial virus (RSV) Fusion (F) polypeptide, wherein the polypeptidecomprises at least two stabilizing mutations in the F1 and/or F2 domainas compared to the RSV F1 and/or F2 domain in a wild-type RSV F protein,wherein at least one of the stabilizing mutations is a mutation of aminoacid residue L at position 203 to I.
 2. The nucleic acid moleculeaccording to claim 1, wherein the RSV F polypeptide further comprises amutation of amino acid residue S at position 215 to P.
 3. The nucleicacid molecule according to claim 1, wherein the RSV F polypeptidecomprises at least one epitope that is specific to the pre-fusionconformation F protein, wherein the at least one epitope is recognizedby a pre-fusion specific monoclonal antibody, comprising a heavy chainCDR1 region of SEQ ID NO: 1, a heavy chain CDR2 region of SEQ ID NO: 2,a heavy chain CDR3 region of SEQ ID NO: 3 and a light chain CDR1 regionof SEQ ID NO: 4, a light chain CDR2 region of SEQ ID NO: 5, and a lightchain CDR3 region of SEQ ID NO: 6 and/or a pre-fusion specificmonoclonal antibody, comprising a heavy chain CDR1 region of SEQ ID NO:7, a heavy chain CDR2 region of SEQ ID NO: 8, a heavy chain CDR3 regionof SEQ ID NO: 9 and a light chain CDR1 region of SEQ ID NO: 10, a lightchain CDR2 region of SEQ ID NO: 67, and a light chain CDR3 region of SEQID NO:
 11. 4. The nucleic acid molecule according to claim 1, whereinthe RSV F polypeptide is trimeric.
 5. The nucleic acid moleculeaccording to claim 1, wherein the RSV F polypeptide comprises atruncated F1 domain, wherein the RSV F polypeptide comprises aheterologous trimerization domain linked to the truncated F1 domain. 6.The nucleic acid molecule according to claim 5, wherein the heterologoustrimerization domain comprises the amino acid sequenceGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 14).
 7. The nucleic acidmolecule according to claim 5, wherein the trimerization domain islinked to the truncated F1 domain at amino acid residue 513 of the RSV Fprotein.
 8. The nucleic acid molecule according to claim 1, wherein theat least one further mutation of the RSV F polypeptide is selected fromthe group consisting of: (a) a mutation of the amino acid residue S atposition 46 to G; (b) a mutation of the amino acid residue N/T atposition 67 to I; (c) a mutation of the amino acid residue L at position83 to M: (d) a mutation of the amino acid residue E at position 92 to D;(e) a mutation of the amino acid residue G at position 184 to N; (f) amutation of the amino acid residue V at position 207 to I: (g) amutation of the amino acid residue D at position 486 to N; and (h) amutation of the amino acid residue E at position 487 to Q, N or I. 9.The nucleic acid molecule according to claim 1, wherein the F1 domainand/or the F2 domain of the RSV F polypeptide are from an RSV A strain.10. The nucleic acid molecule according to claim 1, wherein the F1domain and/or the F2 domain of the RSV F polypeptide are from an RSV Bstrain.
 11. The nucleic acid molecule according to claim 1, wherein theRSV F polypeptide comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO:
 24. 12.The nucleic acid molecule according to claim 1, wherein the nucleic acidmolecule has been codon-optimized for expression in mammalian cells. 13.A vector comprising the nucleic acid molecule according to claim
 1. 14.A composition comprising the nucleic acid molecule according to claim 1.15. A method of inducing an immune response against RSV F protein in asubject in need thereof, the method comprising administering to thesubject the nucleic acid molecule according to claim
 1. 16. Apharmaceutical composition comprising the nucleic acid moleculeaccording to claim 1, and a pharmaceutical carrier.